This invention relates to the fields of pharmaceutical and organic chemistry and provides novel cryptophycin compounds useful as anti-microtubule agents.
Neoplastic diseases, characterized by the proliferation of cells not subject to the normal control of cell growth, are a major cause of death in humans and other mammals. Clinical experience in cancer chemotherapy has demonstrated that new and more effective drugs are desirable to treat these diseases.
The microtubule system of eucaryotic cells is a major component of the cytoskeleton and is a dynamic assembly and disassembly; this is heterodimers of tubulin are polymerized and form microtubule. Microtubules play a key role in the regulation of cell architecture, metabolism, and division. The dynamic state of microtubules is critical to their normal function. With respect to cell division, tubulin is polymerized into microtubules that form the mitotic spindle.
The microtubules are then depolymerized when the mitotic spindle""s use has been fulfilled. Accordingly, agents which disrupt the polymerization or depolymerization of microtubules, and thereby inhibit mitosis, comprise some of the most effective cancer chemotherapeutic agents in clinical use.
Additionally, the compounds claimed herein possess fungicidal properties as well. Further, such agents having the ability to disrupt the microtubule system can be useful for research purposes.
Certain cryptophycin compounds are known in the literature; however, cryptophycin compounds having even greater solubility and stability are desired for most pharmaceutical uses. Further, a broader library of cryptophycin compounds could provide additional treatment options for the patient suffering from cancer. Applicants have now discovered novel compounds which can provide greater aqueous solubility as well as compounds having the ability to disrupt the microtubule system. Compounds of this invention can be useful for the treatment of neoplasms. Such compounds can be prepared using total synthetic methods and are therefore well suited for development as pharmaceutically useful agents.
The presently claimed invention provides novel cryptophycin compounds of Formula I 
Ar is selected from the group consisting of phenyl, any simple unsubstituted aromatic, simple substituted aromatic, substituted heteroaromatic group, unsubstituted heteroaromatic group, heterocyclic, C1-C12 alkyl, C2-C12 alkenyl, C2-C12 alkynyl, NR51R52, COR52, OR53, and Formula Arxe2x80x2 xe2x80x94CH2xe2x80x94CHxe2x95x90CH-phenyl, xe2x80x94S-phenyl, 3-methoxyphenyl, 3,5-dimethoxyphenyl, 4-trifluoromethylphenyl, 4-TBDMSOH2Cxe2x80x94Ph, 4-t-BocHNH2Cxe2x80x94Ph, 3-t-BocHNxe2x80x94Ph, 4xe2x80x94HOOCH2Cxe2x80x94Ph, and 4-HOH2Cxe2x80x94Ph. 
R51 is selected from the group consisting of hydrogen and C1-C3 alkyl;
R52 is selected from the group consisting of hydrogen and C1-C3 alkyl;
R53 is selected from the group consisting of C1-C12 alkyl;
R54 is selected from the group consisting of hydrogen, C1-C6 alkyl, C1-C6alkyl(R57xe2x80x2R57xe2x80x2R57xe2x80x2xe2x80x3), simple unsubstituted aromatic, simple substituted aromatic, heterocyclic, phenyl, halogen, 4-(tert-butyldimethylsiloxy)-benzyltriphenylphosophonium, COOR57, PO3H, SO3H, SO2R58, N(R59)R60, NHOR61, NHCHR61xe2x80x2, CN, NO2, halogen, OR62, CH2(O)R62xe2x80x2, xe2x80x94CH2OC(O)R95, CH2N(R96)R96xe2x80x2, COR100, (C1-C6alkyl)OR100, SR63; 
xe2x80x83and
R95 is selected from the group consisting of xe2x80x94R98NH2;
R96 and R96xe2x80x2 are each independently selected from the group consisting of hydrogen and C1-C6 alkyl, xe2x80x94R97NH2, and xe2x80x94R99 NR99xe2x80x2 R99xe2x80x3;
R97 is selected from the group consisting of C1-C6 alkyl;
R98 is selected from the group consisting of C1-C6 alkyl;
R99 is C1-C6 alkyl;
R99xe2x80x2 and R99xe2x80x3 are each independently selected from the group consisting of hydrogen and C1-C6 alkyl;
R100 is selected from the group consisting of hydrogen, and Si(R101R102R103);
R101 is C1-C6 alkyl;
R102 is C1-C6 alkyl;
R103 is C1-C6 alkyl;
R104 is selected from the group consisting of C(O)C1-C6 alkylN(R106) (R59)R60, C(O)C1-C6 alkylN+, fused bicyclic, and NHR105N(R106) (R59)R60;
R105 is selected from the group consisting of C(O)C1-C6 alkyl, C1-C6 alkyl;
R106 is selected from the group consisting of hydrogen, C1-C6 alkyl, C(O)OR107;
R107 is selected from the group consisting of hydrogen, C1-C6 alkyl, CR108 R109 R110;
R108 is selected from the group consisting of hydrogen and C1-C6 alkyl;
R109 is selected from the group consisting of hydrogen and C1-C6 alkyl;
R110 is selected from the group consisting of hydrogen and C1-C6 alkyl;
R111 is selected from the group consisting of hydrogen, C1-C6 alkyl, and C(O)OR107;
R55 is selected from the group consisting of hydrogen, C1-C6 alkyl, C(R57xe2x80x2R57xe2x80x3R57xe2x80x2xe2x80x3), simple unsubstituted aromatic, simple substituted aromatic, phenyl, COOR57, PO3H, SO3H, SO2R58, NR59R60, NHOR61, NHCHR61xe2x80x2, CN, NO2, halogen, OR62, and SR63;
R56 is selected from the group consisting of hydrogen, C1-C6 alkyl, C(R57xe2x80x2R57xe2x80x3R57xe2x80x2xe2x80x3), simple unsubstituted aromatic, simple substituted aromatic, phenyl, COOR57, PO3H, SO3H, SO2R58, NR59R60, NHOR61, NHCHR61xe2x80x2, (C1-C6)alkylNR59R60, CN, NO2, halogen, OR104 , CR104, OR62, and SR63;
R57 is selected from the group consisting of hydrogen and C1-C12 alkyl;
R57xe2x80x2 is selected from the group consisting of hydrogen, halogen, and C1-C12 alkyl;
R57xe2x80x3 is selected from the group consisting of hydrogen, halogen, and C1-C12 alkyl;
R57xe2x80x2xe2x80x3 is selected from the group consisting of hydrogen, halogen, and C1-C12 alkyl;
R58 is selected from the group consisting of hydrogen and C1-C12 alkyl;
R59 is selected from the group consisting of hydrogen, (C1-C6) alkyl, tert-butoxycarbonyl, carbo-tert-butoxy (t-BOC) and fluorenylmethoxycarbonyl (FMOC);
R60 is selected from the group consisting of hydrogen and (C1-C6) alkyl;
R61 is selected from the group consisting of hydrogen, OR64, CH2NHR65, NHR65xe2x80x2 and fluorenylmethoxycarbonyl (FMOC);
R61xe2x80x2 is selected from the group consisting of hydrogen, OR64, CH2NHR65, NHR65xe2x80x2 and fluorenylmethoxycarbonyl (FMOC);
R62 is selected from hydrogen, and C1-C6 alkyl;
R62xe2x80x2 is selected from hydrogen, OH, OR62, and C1-C6 alkyl;
R63 is selected from hydrogen and C1-C6 alkyl;
R64 is selected from the group consisting of hydrogen, (C1-C6) alkyl, CH2NR66R67;
R65 is selected from the group consisting of hydrogen and C1-C6 alkyl, NH2, and fluorenylmethoxycarbonyl (FMOC);
R65xe2x80x2 is selected from the group consisting of hydrogen and C1-C6 alkyl, NH2, and fluorenylmethoxycarbonyl (FMOC);
R66 is selected from the group consisting of hydrogen and C1-C6 alkyl and fluorenylmethoxycarbonyl (FMOC);
R67 is selected from the group consisting of hydrogen and C1-C6 alkyl;
R1 and R2 are each independently selected from the group consisting of halogen, monalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, phosphate, OR31, SR31, NR31, OH, SH, NR92, R93, NR94, and NH2;
R92, R93, and R94 are each independently selected from the group consisting of C1-C6 alkyl;
provided that one selected from the group consisting of R1 and R2 is selected from the group consisting of OR31, SR31, R31, OH, and SH; or
R1 and R2 may be taken together with C-18 and C-19 to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring, a cyclopropyl ring or mono(C1-C6)alkylphosphate ring; or
R1 and R2 may be taken together to form a second bond between C-18 and C-19;
R3 is a lower alkyl group;
R4 is H or OH;
R5 is H or OH;
R4 and R5 may be taken together to form a second bond between C13 and C14;
R6 is a substituent selected from the group consisting of benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, haloalkoxybenzyl, or dihaloalkyoxybenzyl group, B-ring heteroaromatic, substituted heteroaromatic, B-ring (C1-C6)alkyl, (C3-C8)cycloalkyl, substituted C3-C8 cycloalkyl, substituted (C1-C6)alkyl, a group of the formula IIIxe2x80x2
xe2x80x83and a group of the formula IIIxe2x80x3: 
R7 is selected from the group consisting of NR51R52, R53NR51R52, OR53, H and a lower alkyl group; R51 and R52 are independently selected from the group consisting of C1-C3 alkyl; R53 is C1-C3 alkyl;
R8 is H or a lower alkyl group; or
R7 and R8 can form a cyclopropyl ring;
R9 is selected from the group consisting of H, a lower alkyl group, unsaturated lower alkyl, lower alkyl-C3-C5 cycloalkyl, and benzyl;
R10 is H or a lower alkyl group;
R11 is selected from the group consisting of hydrogen, OH, lower alkyl group, substituted phenyl, benzyl, substituted benzyl and phenyl;
R14 is selected from the group consisting of hydrogen and lower alkyl;
R15, R16, and R17 are each independently selected from the group consisting of hydrogen, OR18, halo, NR18xe2x80x2R19xe2x80x2, NO2, OPO3H2, OR19phenyl, SCH2phenyl, CONH2, CO2H, PO3H2, SO2R23, and ZZ;
R18 is selected from the group consisting of hydrogen, aryl, C1-C6 alkyl, C(O)R90 and fluorenylmethoxycarbonyl (FMOC);
R18xe2x80x2 is selected from the group consisting of hydrogen, (C1-C6)alkyl and C(O)R90xe2x80x2;
R19 is C1-C6 alkyl, C(O)R90xe2x80x3 and fluorenylmethoxycarbonyl (FMOC);
R19xe2x80x2 is selected from the group consisting of hydrogen, (C1-C6)alkyl, and C(O)R90xe2x80x2xe2x80x3;
R90, R90xe2x80x2, R90xe2x80x3, and R90xe2x80x2xe2x80x3 are each independently selected from the group consisting of hydrogen, (C1-C6)alkyl, OR91 and aryl;
R91xe2x80x2 is selected from the group consisting of (C1-C6)alkyl, aryl, and hydrogen;
R23 is selected from the group consisting of hydrogen and (C1-C3) alkyl;
R30 is hydrogen or C1-C6 alkyl; or
R30 may be taken together with the N at C-11 to form a three to seven membered cyclic ring;
R31 is selected from the group consisting of P, S, (C1-C12) alkyl, B, R32 and Si;
R32 is selected from the group consisting of amino acid, carbohydrate, amino sugar, (saccharide)q, C(O)R33, and 
R33 is selected from the group consisting of R37R38, R38, R37N (R20xe2x80x2)R38, R37N(R20xe2x80x2) (C1-C6)alkylC(O)R38, R37N(R20xe2x80x2) C(O)R38, R37O(C1-C6)alkylO(C1-C6)alkylO(C1-C6)alkylO(C1-C6)alkyl and R37(N R18xe2x80x2R20xe2x80x2)R38;
R20xe2x80x2 is selected from the group consisting of hydrogen, C1-C6 alkyl, and xe2x80x94CO2R21;
R21xe2x80x2 is selected from the group consisting of hydrogen and (C1-C6)alkyl;
R34 is (C1-C4)alkyl;
R35 is hydrogen or (C1-C3)alkyl;
R36 is hydrogen, OH, halo, (C1-C3)alkyl, OR34, NO2, NH2 and heteroaromatic;
R37 is (C1-C6)alkyl;
R38 is COOR39, 
xe2x80x83NH2, (N R18xe2x80x2R20xe2x80x2), heterocyclic, heteroaromatic, OH, (C1-C6)alkyl, and amino acid;
R39 is H or (C1-C6)alkyl;
R40, R41, and R42 are each independently selected from the group consisting of hydrogen, OR43, halo, NH2, NO2, OPO(OR46)2, xe2x80x94OR44phenyl, and R45;
R43 is C1-C6 alkyl;
R44 is C1-C6 alkyl;
R45 is selected from the group consisting of a unsubstituted simple aromatic group and a substituted simple aromatic group;
R46 is selected from the group consisting of H, Na, (C1-C6)alkyl and xe2x80x94C(CH3)3;
R50 is hydrogen or 
n is 0, 1, or 2;
m is 0, 1, or 2;
p is 0, 1, or 2;
q is 2, 3, or 4;
X is selected from the group consisting of O, C, S, NH and alkylamino;
Y is selected from the group consisting of C, O, NH, S, SO, SO2 and alkylamino;
Z is selected from the group consisting of xe2x80x94(CH2)nxe2x80x94, xe2x80x94(CH2)pxe2x80x94Oxe2x80x94(CH2)mxe2x80x94 and (C3-C5) cycloalkyl;
ZZ is selected from the group consisting of a simple unsubstituted aromatic group and a simple substituted aromatic group; or
a pharmaceutically acceptable salt or solvate thereof;
provided that if R1 is selected from the group consisting of halogen, OH, OR31, SH, amino, monoalkylamino, dialkylamino, trialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate and phosphate, and R2 is selected from the group consisting of OH, NH2, NR31 and SH or R1 and R2 together form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring, a cyclopropyl ring, or monoalkylphosphate ring, or R1 and R2 together form a second bond; R3 is lower alkyl; R4 and R5 are H or R4 and R5 taken together form double bond between C13 and C14; R6 is benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, halohydroxybenzyl, or dihalohydroxybenzyl; R7, R8, R9, and R10 are each independently H or a lower alkyl group; and X and Y are each independently O, NH or alkylamino and R50 is 
xe2x80x83and R11 is hydrogen; then
Ar is not selected from the group consisting of C1-C12alkyl, C1-C12alkynyl, phenyl, simple unsubstituted aromatic, substituted aromatic, unsubstituted heteroaromatic, and substituted heteroaromatic; or
if Ar is selected from the group consisting of C1-C12alkyl, C1-C12alkynyl, phenyl, simple unsubstituted aromatic, substituted aromatic, unsubstituted heteroaromatic, and substituted heteroaromatic; and R50 is 
xe2x80x83and R11 is hydrogen; then
R2 is selected from the group consisting of halogen, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, phosphate, OR31 and SR31; or
if R1 is selected from the group consisting of halogen, OH, OR31, SH, amino, monoalkylamino, dialkylamino, trialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate and phosphate, and R2 is selected from the group consisting of OH, NH2, NR31 and SH or R1 and R2 together form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring, a cyclopropyl ring, or monoalkylphosphate ring, or R1 and R2 together form a second bond and R3 is lower alkyl and R4 is H and R5 is H and R50 is hydrogen and R11 is hydrogen; then
Ar is not selected from the group consisting of C1-C12alkyl, C1-C12alkynyl, phenyl, simple unsubstituted aromatic, substituted aromatic, and heteroaromatic; or
if R1 is selected from the group consisting of halogen, OH, OR31, SH, amino, monoalkylamino, dialkylamino, trialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate and phosphate, and R2 is selected from the group consisting of OH, NH2, NR31 and SH or R1 and R2 together form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring, a cyclopropyl ring, or monoalkylphosphate ring, or R1 and R2 together form a second bond and R3 is lower alkyl and R4 is H and R5 is H and R50 is hydrogen and R11 is hydrogen; or R50 is selected from the group consisting of 
xe2x80x83and hydrogen; and R11 is selected from the group consisting of OH, lower alkyl group, substituted phenyl, benzyl, substituted benzyl and phenyl;
then
Ar is not selected from the group consisting of C1-C12alkyl, C1-C12alkynyl, phenyl, simple unsubstituted aromatic, substituted aromatic, substituted heteroaromatic and unsubstituted heteroaromatic; or
if R3 is lower alkyl; R4 and R5 are H or R4 and R5 taken together form a double bond between C13 and C14; R6 is benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, dihalohydroxybenzyl, halohydroxybenzyl, or dihalohydroxybenzyl; R7, R8, R9, and R10 are each independently H or a lower alkyl group; and X and Y are each independently O, NH or alkylamino; and Ar is of the formula Arxe2x80x2 and
one of R54 , R55, R56 is selected from the group consisting of alkyl or halogen; and
R1 and R2 may be taken together to form an epoxide ring, an aziridine ring, an episulfide ring, a sulfate ring, a cyclopropyl ring or monoalkylphosphate ring; or R1 and R2 may be taken together to form a second bond between C18 and C19; or R2 is selected from the group consisting of OH and SH;
then at least two of R54, R55, and R56 must be selected from the group consisting of C1-C6 alkyl, simple aromatic, phenyl, COOR57, PO3H, SO3H, SO2R58, NR59R60, NHOR61, NHCHR61xe2x80x2, CN, NO2, halogen, OR62, and SR63; or
if R3 is lower alkyl; R4 and R5 are H or R4 and R5 taken together form a double bond between C13 and C14; R6 is B-ring heteroaromatic, substituted heteroaromatic, B-ring (C1-C6)alkyl, (C3-C8)cycloalkyl, substituted C3-C8 cycloalkyl, substituted (C1-C6)alkyl, a group of the formula IIIxe2x80x2
xe2x80x83and a group of the formula IIIxe2x80x3: 
xe2x80x83and
X and Y are each independently O, NH or alkylamino; then
Ar is not selected from the group consisting of phenyl or any simple unsubstituted or substituted aromatic or heteroaromatic group, C1-C12 alkyl, and C1-C12 alkynyl.
The present invention provides pharmaceutical formulations, a method for disrupting a microtubulin system using an effective amount of a compound of Formula I, a method for inhibiting the proliferation of mammalian cells comprising administering an effective amount of a compound of Formula I, and a method for treating neoplasia in a mammal comprising administering an effective amount of a compound of Formula I. Also, provided is a method for controlling a mycotic infection comprising administering to an animal infected with or susceptible to infection with a fungi, an antifungally effective amount of a compound of Formula I.
As used herein, the term xe2x80x9csimple alkylxe2x80x9d shall refer to C1-C7 alkyl wherein the alkyl may be saturated, unsaturated, branched, or straight chain. Examples include, but are in no way limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, propenyl, ethenyl, sec-butyl, n-pentyl, isobutyl, tert-butyl, sec-butyl, methylated butyl groups, pentyl, tert pentyl, sec-pentyl, methylated pentyl groups and the like. The term xe2x80x9calkenylxe2x80x9d refers to an alkyl group, as defined above, having from one to three double bonds. The term xe2x80x9calkynylxe2x80x9d refers to an alkyl group, as defined above, having at least one triple bond. It is especially preferred that alkynyl has only one triple bond. The term C1-Cnxe2x80x2 alkyl; wherein nxe2x80x2 is an integer from 2 to 12 means an alkyl group having from one to the indicated number of carbon atoms. The C1-Cnxe2x80x2 alkyl can be straight or branched chain.
As used herein, the term xe2x80x9cB-ring C1-C6 alkylxe2x80x9d refers to saturated, unsaturated, branched and straight chain alkyl wherein the B-ring C1-C6alkyl group may include up to three (3) non-carbon substituents. Such non-carbon substituents are most preferredly selected from the group consisting of OH, SCH2phenyl, NH2, CO, CONH2, CO2H, PO3H2, SO2R21 wherein R21 is selected from hydrogen and C1-C3alkyl.
As used herein the term xe2x80x9camino acidxe2x80x9d means an organic acid containing an amino group. The term includes both naturally occurring and synthetic amino acids, therefore, the amino group can be, but is not required to be, attached to the carbon next to the acid. The amino acid substituent is attached to the parent molecule via the organic acid functionality.
As used herein, the term xe2x80x9ccarbohydratexe2x80x9d refers to a class of substituents made up of carbon, hydrogen, and oxygen wherein hydrogen and oxygen are in the same proportions as in water or nearly the proportions as water. The term xe2x80x9ccarbohydratexe2x80x9d further refers to an aldehyde or ketone alcohol or a compound which on hydrolysis produces an aldehyde or ketone. The term xe2x80x9ccarbohydratexe2x80x9d is as commonly understood by the skilled artisan. For example, the term refers to, but is in no way limited to, C12H22O11 and C6H10O5.
As used herein, the term xe2x80x9camino sugarxe2x80x9d refers to a carbohydrate group containing from one to three amino substituents at any available position on the carbohydrate molecule.
As used herein, the term xe2x80x9csaccharidexe2x80x9d refers to carbohydrate subunits to form disaccharides or polysaccharides. The term means for example, but in no way limited to, lactose, maltose, sucrose, fructose, starch, and the like.
As used herein, the term xe2x80x9csubstituted phenylxe2x80x9d shall refer to a phenyl group with from one to three non-hydrogen substituents which may be independently selected from the group consisting of simple alkyl, Cl, Br, F, and I.
As used herein, the term xe2x80x9csubstituted benzylxe2x80x9d shall refer to a benzyl group with from one to three non-hydrogen substituents which may be independently selected from the group consisting of simply alkyl, Cl, Br, F, and I wherein such substituents may be attached at any available carbon atom. Some preferred substituted benzyls have been described as well. As used herein, the term xe2x80x9calkoxybenzylxe2x80x9d refers to a benzyl group having an alkoxy substituent at any available position on the benzyl ring The alkoxy group is most preferably -) (C1-C3) alkyl. Methoxy is especially preferred. Accordingly, the term xe2x80x9chaloalkoxybenzylxe2x80x9d refers to a benzyl group having a halo substituent in addition to an alkoxy substituent. Each halo or alkoxy group is substituted at any available carbon. Similarly, xe2x80x9chalohydroxybenzylxe2x80x9d refers to a hydroxy substituted benzyl group that also has a halo substituent at any available carbon on the benzyl ring. Finally, the term xe2x80x9cdihaloalkoxybenzylxe2x80x9d refers to an alkoxy substituted benzyl which additionally has two halo substituents each independently substituted at any available carbon on the benzyl ring.
As used herein xe2x80x9cB-ring heteroaromatic groupxe2x80x9d refers to aromatic rings which contain one or more non-carbon substituent selected from the group consisting of oxygen, nitrogen, and sulfur. Especially preferred B-ring heterocyclic groups are selected from, but not limited to, the group consisting of: 
R20 is selected from hydrogen and C1-C6 alkyl.
It is especially preferred that xe2x80x9cB-ring heteroaromatic groupxe2x80x9d refers to a substituent selected from the group consisting of: 
xe2x80x9cSubstituted aromaticxe2x80x9d refers to a group substituted with from one to three substituents selected from the group consisting of simple alkyl and halo.
As used herein xe2x80x9ccycloalkylxe2x80x9d refers to a saturated C3-C8 cycloalkyl group wherein such group may include from zero to three substituents selected from the group consisting of C1-C3 alkyl, halo, and OR22 wherein R22 is selected from hydrogen and C1-C3 alkyl. Such substituents may be attached at any available carbon atom. It is especially preferred that cycloalkyl refers to substituted or unsubstituted cyclohexyl.
As used herein xe2x80x9cLower alkoxyl groupxe2x80x9d means any alkyl group of one to five carbon atoms bonded to an oxygen atom. As used herein xe2x80x9clower alkyl groupxe2x80x9d means an alkyl group of one to six carbons and includes linear and non-linear hydrocarbon chains, including for example, but not limited to methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, sec-butyl, methylated butyl groups, pentyl, tert pentyl, sec-pentyl, and methylated pentyl groups. As used herein, the term xe2x80x9cunsaturated lower alkyl groupxe2x80x9d and xe2x80x9csaturated lower alkyl groupxe2x80x9d shall have the meaning that the artisan commonly associates with the terms unsaturated and saturated. The term xe2x80x9clower alkylxe2x80x9d shall refer to both saturated and unsaturated lower alkyl groups. (For example a saturated group has no double or triple bonds). As used herein xe2x80x9callylically substituted alkenexe2x80x9d means any alkene having from two to seven carbon atoms which contains an alkyl substitution on it.
As used herein xe2x80x9cepoxide ringxe2x80x9d means a three-membered ring whose backbone consists of two carbons and an oxygen atom. As used herein, xe2x80x9caziridine ringxe2x80x9d means a three-membered ring whose backbone consists of two carbon atoms and a nitrogen atom. As used herein xe2x80x9csulfide ringxe2x80x9d means a three-membered ring whose backbone consists of two carbon atoms and a sulfur atom. As used herein xe2x80x9cepisulfide ringxe2x80x9d means a three-membered ring whose backbone consists of two carbon and a sulfur atom. As used herein xe2x80x9csulfate groupxe2x80x9d means a five membered ring consisting of a carbon-carbon-oxygen-sulfur-oxygen backbone with two additional oxygen atoms connected to the sulfur atom. As used herein, xe2x80x9cmonalkylphosphate ringxe2x80x9d means a five membered ring consisting of a carbon-carbon-oxygen-phosphorous-oxygen backbone with two additional oxygen atoms, one of which bears a lower alkyl group, connected to the phosphorous atom.
As used herein, xe2x80x9csimple unsubstituted aromatic groupxe2x80x9d refers to common aromatic rings having 4n+2 electrons in a moncyclic or bicyclic conjugated system, for example, but not limited to: furyl, pyrrolyl, thienyl, pyridyl, and the like, or a bicyclic conjugated system, for example, but not limited to, indolyl or naphthyl.
As used herein xe2x80x9csimple substituted aromatic groupxe2x80x9d refers to a simple aromatic ring substituted with a single group selected from the group consisting of halogen and lower alkyl group.
As used herein, xe2x80x9cheteroaromaticxe2x80x9d refers to aromatic rings which contain one or more non-carbon atoms selected from the group consisting of oxygen, nitrogen, and sulfur. Most preferred heteroaromatic rings have from three to eight members in the ring. An especially preferred group of heteroaromatic rings have from three to six members. It is particularly preferred that the heteroaromatic group will have from one to three non-carbon atoms in the ring. A five member ring containing one oxygen atom is one preferred heteroaromatic group; however, the term is in no way limited to this group.
As used herein xe2x80x9cheterocyclicxe2x80x9d refers to cyclic rings which contain one or more non-carbon atoms selected from the group consisting of oxygen, nitrogen, and sulfur. The heterocyclic rings may be saturated or unsaturated. Further, the heterocyclic rings may be fused with one another to form a bicyclic or tricyclic system. For example, but not limited to a five membered ring having two double bonds or a five membered ring having one double bond. The heterocyclic rings may be unsubstituted or may have from one to three substituents selected from the group consisting of C1-C6alkyl, carbonyl, halogen, and OR62. Most preferred heterocyclic rings have from three to eight members in the ring. An especially preferred group of heterocyclic rings have from three to six members. It is particularly preferred that the heterocyclic group will have from one to three non-carbon atoms. One preferred heterocyclic ring is a five membered ring having one nitrogen, one sulfur, one methyl substituent, and two double bonds.
A preferred heterocyclic group includes but is not limited to: 
xe2x80x9cSubstituted heteroaromaticxe2x80x9d refers to a group substituted with from one to three substituents selected from the group consisting of simple alkyl and halo.
As used herein, xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d refers to those members of the group on the periodic table historically known as halogens. Methods of halogenation include, but are not limited to, the addition of hydrogen halides, substitution at high temperature, photohalogenation, etc., and such methods are known to the skilled artisan. Especially preferred halogens include, but are in no way limited to: chloro, fluoro, and bromo.
As used herein, the term xe2x80x9carylxe2x80x9d has the meaning commonly associated with the term by the artisan. Thus, the term means an organic radical derived from an aromatic hydrocarbon by the removal of one hydrogen atom. For example, but not limited to, phenyl, tolyl, salicyl, and the like.
As used herein, the term xe2x80x9cmammalxe2x80x9d shall refer to the Mammalia class of higher vertebrates. The term xe2x80x9canimalxe2x80x9d shall include, but is not limited to, mammals, reptiles, amphibians, and fish. The term xe2x80x9cmammalxe2x80x9d includes, but is not limited to, a human. The term xe2x80x9ctreatingxe2x80x9d as used herein includes prophylaxis of the named condition or amelioration or elimination of the condition once it has been established. The cryptophycin compounds claimed herein can be useful for veterinary health purposes as well as for the treatment of a human patient.
When the desired R6 substituent in the compound contains an amine, then the amine substituent of the R6 group must be protected using an amino protecting group. The artisan can readily select an appropriate amino protecting group using guidance from standard works, including, for example, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, (London and New York, 1973); Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981).
As used herein, the term xe2x80x9cderivatizingxe2x80x9d refers to standard chemical transformations known to the artisan which give access to desired compounds of this invention.
The presently claimed processes provide a means for preparing totally synthetic cryptophycin compounds. Conveniently, commercially available amino acids can be cyclized into the cryptophycin molecule.
Many of the known cryptophycin compounds have promising antitumor activity; however, poor aqueous solubility can present issues during intravenous administration of drug. Such issues are related to the use of solubilizing surfactants, such a Cremophor, which may possess inherent toxicity. The present invention provides new cryptophycin compounds having antitumor activity as well as greater aqueous solubility properties. Such compounds have desired solubility characteristics as well as acceptable potency.
The processes to prepare the compounds of this invention most preferably are completed in the presence of a solvent. The skilled artisan can select appropriate solvents using standard methodologies. Suitable inert organic solvents include those known to the skilled artisan, for example, but not limited to, tetrahydrofuran (THF) and dimethylformamide (DMF). DMF is especially preferred. Aqueous based solvents may be appropriate for some of the processes utilized herein. The pH of such aqueous solvent s may be adjusted as desired to facilitate the process.
Some typical compounds of this invention are provided in tabular form; however, such named compounds are not intended to limit the scope of this invention in any way.
Additional preferred compounds are, for example, but not limited to, those named above in Table I wherein the positions of the adjacent Cl and OR groups are traded. For example, the same R substituents named above wherein the base structure is as follows: 
Additional compounds of interest are as named by Table I; however, Ar is Arxe2x80x2 instead of phenyl and R54 is OCH3 at the para position of Arxe2x80x2, while R55 and R56 are each hydrogen.
Further compounds of interest are as named by Table I; however, an NH group replaces O at the Y position of the molecule. For example, one compound in this series is: 
Further preferred compounds are, for example, but not limited to, those named above in Table I as well as by the compounds named by the traded positions of the adjacent Cl and OR; wherein each named compound has a single methyl group in place of the gem dimethyl group at position 6 of the cryptophycin ring. See, for example, 23 below.
Especially preferred compounds of this invention have a dimethyl group at position 6 of the cryptophycin ring.
A preferred compound of this invention is as presented in Table I wherein Ar is Arxe2x80x2; only one of R54,R55, R56 is OCH3; R9 and R10 are each methyl.
Generally known silylating agents are employed in the processes for making compounds of this invention. See for example, Calvin, E. W., xe2x80x9cSilicon Reagents in Organic Synthesisxe2x80x9d, Academic Press, (London, 1988). Particularly useful silylating agents include xe2x80x9ctri-lower alkyl silylxe2x80x9d agents, the term of which contemplates truisopropylsilyl, trimethylysilyl and triethylsilyl, trimethylsilyl halides, silylated ureas such as bis(trimethylsilyl)urea (BSU) and silylated amides such as N,O-bis(trimethylsilyl)acetamide (BSA). Of these, BSA is preferred.
Some preferred characteristics of this invention are set forth in the following tabular form wherein the features may be independently selected to provide preferred embodiments of this invention. The invention is in no way limited to the features described below:
A) R8 is ethyl, propyl, isopropyl, butyl, isobutyl or isopentyl;
B) R7 is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl, or isopentyl;
C) R7 is H, R8 is methyl, R3 is methyl, and X and Y are not both O;
D) R3 is ethyl, propyl, isopropyl, butyl, isobutyl, pentyl or isopentyl;
E) R9 is methyl, ethyl, propyl, butyl, isobutyl, pentyl, or isopentyl;
F) R10 is methyl, ethyl, propyl, butyl, isobutyl, pentyl, or isopentyl;
G) a cryptophycin compound wherein at least one of the groups selected from the group consisting of C-3, C-6, C-7, C-10, C-16, C-17, and C-18 has R stereochemistry (numbering as set forth in Formula I supra.);
H) a cryptophycin compound wherein at least one of the groups selected from the group consisting of C-3, C-6, C-7, C-10, C-16, C-17, and C-18 has S stereochemistry (numbering as set forth in Formula I supra.);
I) Arxe2x80x2 is phenyl with substituent selected from the group consisting of NR59R60, NHOR61 and NHCHR61xe2x80x2;
J) a compound wherein Y is selected from the group consisting of alkylamino, NH, and O;
K) a compound wherein Y is O, R7 and R10 are each hydrogen; R9 is lower alkyl; and R1 is halo;
L) R7, R8 are each methyl;
M) R7 is hydrogen;
N) R2 is a glycinate;
O) R2 is an acylate;
P) R1 and R2 form an epoxide ring;
Q) R6 is selected from the group consisting of R6 is benzyl, hydroxybenzyl, alkoxybenzyl, halohydroxybenzyl, and dihalohydroxybenzyl;
R) R4 and R5 form a double bond;
S) n is 0; R6 is substituted benzyl wherein one substituent is a halogen and one is an OR12 group wherein R12 is lower alkyl;
T) a compound of Formula I is used for disruption of a microtubulin system;
U) a compound of Formula I is used as a antineoplastic agent;
V) a compound of Formula I is used for the treatment of cancer in a mammal;
W) a compound of Formula Isis used as an antifungal agent;
X) R6 is Formula IIIxe2x80x2 and is para hydroxy substituted;
R6 is selected from the group consisting 
Z) Z is xe2x80x94(CH2)nxe2x80x94 wherein n is 0;
AA) Z is xe2x80x94(CH2)nxe2x80x94 wherein n is 2;
BB) Z is xe2x80x94(CH2)nxe2x80x94 wherein n is 1;
CC) at least one of R15, R16, and R17 is selected from the group consisting of SCH2phenyl, NH2, CO, CONH2, CO2H, PO3H2, and SO2R21; wherein R21 is selected from hydrogen and C1-C3 alkyl;
DD) Ar is phenyl;
EE) Ar is phenyl substituted with one or two from the group consisting of OH, OCH3, halo, and methyl;
FF) R2 is selected from the group consisting of halogen, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, phosphate, OR31R32, and SR31R32;
GG) R6 has a Z wherein the first carbon of the Z group is 
xe2x80x83with respect to the point of attachment to the cryptophycin molecule;
a compound of Formula I is used for the treatment of fungal infection;
II) R50 is 
JJ) R11 is hydrogen;
KK) R4 and R5 are each hydrogen;
LL) Ar is para ethyl substituted phenyl;
MM) Ar is para methyl substituted phenyl;
NN) Y is NH;
OO) R3 is methyl;
PP) R6 is selected from the group consisting of: 
The present invention provides a method of alleviating a pathological condition caused by hyperproliferating mammalian cells comprising administering to a subject an effective amount of a pharmaceutical or veterinary composition disclosed herein to inhibit proliferation of the cells. In a preferred embodiment of this invention, the method further comprises administering to the subject at least one additional therapy directed to alleviating the pathological condition. In a preferred embodiment of the present invention, the pathological condition is characterized by the formation of neoplasms.
In a further preferred embodiment of the present invention, the neoplasms are selected from the group consisting of mammary, small-cell lung, non-small-cell lung, colorectal, leukemia, melanoma, pancreatic adenocarcinoma, central nervous system (CNS), ovarian, prostate, sarcoma of soft tissue or bone, head and neck, gastric which includes pancreatic and esophageal, stomach, myeloma, bladder, renal, neuroendocrine which includes thyroid and non-Hodgkins disease and Hodgkin""s disease neoplasms.
As used herein xe2x80x9cneoplasticxe2x80x9d refers to a neoplasm, which is an abnormal growth, such growth occurring because of a proliferation of cells not subject to the usual limitations of growth. As used herein, xe2x80x9canti-neoplastic agentxe2x80x9d is any compound, composition, admixture, co-mixture, or blend which inhibits, eliminates, retards, or reverses the neoplastic phenotype of a cell.
Anti-mitotic agents may be classified into three groups on the basis of their molecular mechanism of action.
The first group consists of agents, including colchicine and colcemid, which inhibit the formation of microtubules by sequestering tubulin. The second group consists of agents, including vinblastine and vincristine, which induce the formation of paracrystalline aggregates of tubulin. Vinblastine and vincristine are well known anticancer drugs: their action of disrupting mitotic spindle microtubules preferentially inhibits hyperproliferative cells. The third group consist of agents, including taxol, which promote the polymerization of tubuline and thus stabilizes microtubules.
The exhibition of drug resistance and multiple-drug resistance phenotype by many tumor cells and the clinically proven mode of action of antimicrotubule agents against neoplastic cells necessitates the development of antimicrotubule agents cytotoxic to non-drug resistance neoplastic cells as well as cytotoxic to neoplastic cells with a drug resistant phenotype.
Chemotherapy, surgery, radiation therapy, therapy with biological response modifiers, and immunotherapy are currently used in the treatment of cancer. Each mode of therapy has specific indications which are known to those of ordinary skill in the art, and one or all may be employed in an attempt to achieve total destruction of neoplastic cells. Moreover, combination chemotherapy, chemotherapy utilizing compounds of Formula I in combination with other neoplastic agents, is also provided by the subject invention as combination therapy is generally more effective than the use of a single anti-neoplastic agent. Thus, a further aspect of the present invention provides compositions containing a therapeutically effective amount of at least one compound of Formula I, including the non-toxic addition salts thereof, which serve to provide the above recited benefits. Such compositions can also be provided together with physiologically tolerable liquid, gel, or solid carriers, diluents, adjuvants and excipients. Such carriers, adjuvants, and excipients may be found in the U.S. Pharmacopoeia, Vol. XXII and National Formulary Vol. XVII, U.S. Pharmacopoeia Convention, Inc., Rockville, Md. (1989). Additional modes of treatment are provided in AHFS Drug Information, 1993 e. By the American Hospital Formulary Service, pp. 522-660. Each of these references are well known and readily available to the skilled artisan.
The present invention further provides a pharmaceutical composition used to treat neoplastic disease containing at least one compound of Formula I and at least one additional anti-neoplastic agent. Anti-neoplastic agents which may be utilized in combination with Formula I compounds include those provided in the Merck Index 11, pp 16-17, Merck and Co., Inc. (1989). The Merck Index is widely recognized and readily available to the skilled artisan.
In a further embodiment of this invention, antineoplastic agents may be antimetabolite which may include but are in no way limited to those selected from the group consisting of methotrexate, 5-fluorouracil, 6-mercaptopurine, cytosine, arabinoside, hydroxyurea, and 2-chlorodeoxyadenosine. In another embodiment of the present invention, the anti-neoplastic agents contemplated are alkylating agents which may include but are in no way limited to those selected from the group consisting of cyclophosphamide, mephalan, busulfan, paraplatin, chlorambucil, and nitrogen mustard. In a further embodiment, the anti-neoplastic agents are plant alkaloids which may include but are in no way limited to those selected from the group consisting of vincristine, vinblastine, taxol, and etoposide. In a further embodiment, the anti-neoplastic agents contemplated are antibiotics which may include, but are in no way limited to those selected from the group consisting of doxorubicin, daunorubicin, mitomycin C, and bleomycin. In a further embodiment, the anti-neoplastic agents contemplated are hormone which may include, but are in no way limited to those selected from the group consisting of calusterone, diomostavolone, propionate, epitiostanol, mepitiostane, testolactone, tamoxifen, polyestradiol phosphate, megesterol acetate, flutamide, nilutamide, and trilotane.
In a further embodiment, the anti-neoplastic agents contemplated include enzymes which may include, but are in no way limited tot hose selected from the group consisting of L-Asparginase and aminoacridine derivatives such as, but not limited to, amsacrine. Additional anti-neoplastic agents include those provided by Skeel, Roland T., xe2x80x9cAntineoplastic Drugs and Biologic Response Modifier: Classification, Use and Toxicity of Clinically Useful Agents"" Handbook of Cancer Chemotherapy (3rd ed.), Little Brown and Co. (1991).
These compounds and compositions can be administered to mammals for veterinary use. For example, domestic animals can be treated in much the same way as a human clinical patient. In general, the dosage required for therapeutic effect will vary according to the type of use, mode of administration, as well as the particularized requirements of the individual hosts. Typically, dosages will range from about 0.001 to 1000 mg/kg, and more usually 0.01 to 10 mg/kg of the host body weight. Alternatively, dosages within these ranges can be administered by constant infusion over an extended period of time, usually exceeding 24 hours, until the desired therapeutic benefits are obtained. Indeed, drug dosage, as well as route of administration, must be selected on the basis of relative effectiveness, relative toxicity, growth characteristics of tumor and effect of Formula I compound on cell cycle, drug pharmacokinetics, age, sex, physical condition of the patient and prior treatment, which can be determined by the skilled artisan.
The compound of Formula I, with or without additional anti-neoplastic agents, may be formulated into therapeutic compositions as natural or salt forms. Pharmaceutically acceptable non-toxic salts include base addition salts which may be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. Such salts may also be formed as acid addition salts with any free cationic groups and will generally be formed with inorganic acids such as for example, hydrochloric or phosphoric acids or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Additional excipients which further the invention are provided to the skilled artisan for example in the U.S. Pharmacopoeia. 
The suitability of particular carriers for inclusion in a given therapeutic composition depends on the preferred route of administration. For example, anti-neoplastic compositions may be formulated for oral administration. Such compositions are typically prepared as liquid solution or suspensions or in solid forms. Oral formulation usually include such additives as binders, fillers, carriers, preservatives, stabilizing agents, emulsifiers, buffers, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations, or powders, and typically contain 1% to 95% of active ingredient. More preferably, the composition contains from about 2% to about 70% active ingredient.
Compositions of the present invention may be prepared as injectables, either as liquid solutions, suspensions, or emulsions; solid forms suitable for solution in or suspension in liquid prior to injection. Such injectables may be administered subcutaneously, intravenously, intraperitoneally, intramuscularly, intrathecally, or intrapleurally. The active ingredient or ingredients are often mixed with diluents, carriers, or excipients which are physiologically tolerable and compatible with the active ingredient(s). Suitable diluents and excipients are for example, water, saline, dextrose, glycerol, or the like and combinations thereof. In addition, if desired, the compositions may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, stabilizing or pH buffering agents.
The invention further provides methods for using Formula I compounds to inhibit the proliferation of mammalian cells by contacting these cells with a Formula I compound in an amount sufficient to inhibit the proliferation of the mammalian cell. A preferred embodiment is a method to inhibit the proliferation of hyperproliferative mammalian cells. For purposes of this invention xe2x80x9chyperproliferative mammalian cellsxe2x80x9d are mammalian cells which are not subject to the characteristic limitations of growth (programmed cell death for example). A further preferred embodiment is when the mammalian cell is human. The invention further provides contacting the mammalian cell with at least one Formula I compound and at least one anti-neoplastic agent. The types of anti-neoplastic agents contemplated are discussed supra.
The invention further provides methods for using a compound of Formula I to inhibit the proliferation of hyperproliferative cells with drug-resistant phenotypes, including those with multiple drug-resistant phenotypes, by contacting said cell with a compound of Formula I in an amount sufficient to inhibit the proliferation of a hyperproliferative mammalian cell. A preferred embodiment is when the mammalian cell is human. The invention further provides contacting a Formula I compound and at least one additional anti-neoplastic agent, discussed supra.
The invention provides a method for alleviating pathological conditions caused by hyperproliferating mammalian cells for example, neoplasia, by administering to a subject an effective amount of a pharmaceutical composition containing Formula I compound to inhibit the proliferation of the hyperproliferating cells. As used herein xe2x80x9cpathological conditionxe2x80x9d refers to any pathology arising from the proliferation of mammalian cells that are not subject to the normal limitations of growth. Such proliferation of cells may be due to neoplasms as discussed supra.
In a further preferred embodiment the neoplastic cells are human. The present invention provides methods of alleviating such pathological conditions utilizing a compound of Formula I in combination with other therapies, as well as other anti-neoplastic agents.
The effectiveness of the claimed compound can be assessed using standard methods know to the skilled artisan.
Examples of such methods are as follows:
Compounds of this invention have been found to be useful against pathogenic fungi. For example, the usefulness for treating Cryptococcus neoformans can be illustrated with test results against Cryptococcus neoformans employing yeast nitrogen base dextrose agar medium. In carrying out the assay, a compound of this invention is solubiized in dimethyl sulfoxide supplemented with Tween 20. Two fold dilutions are made with sterile distilled water/10 percent DMSO to obtain final drug concentrations in the agar dilution assay plates ranging from 0.008 xcexcg/ml to 16.0 xcexcg/ml against an expanded panel of 84 Cryptococcus neoformans strains. The minimum inhibitory concentration against the panel of 84 Cryptococcus neoformans isolates is determined to illustrate the desired antifungal activity.
The compounds are screened for minimum inhibitory concentrations against KB, a human nasopharyngeal carcinoma cell line, LoVo, a human colorectal adenocarcinoma cell line using The Corbett assay, see Corbett, T. H. et al. Cytotoxic Anticancer Drugs: Models and Concepts for Drug Discovery and Development, pp 35-87, Kluwer Academic Publishers: Norwell, 1992. See also, Valeriote, et al. Discovery and Development of Anticancer Agents; Kluwer Academic Publisher, Norwell, 1993 is used for the evaluation of compounds.
The most active compounds are further evaluated for cytotoxicity against four different cell types, for example a murine leukemia, a murine solid tumor, a human solid tumor, and a low malignancy fibroblast using the Corbett assay.
The compounds are further evaluated against a broad spectrum of murine and human tumors implanted in mice, including drug resistant tumors.
Tumor burden (T/C) (mean tumor burden in treated animals versus mean tumor burden in untreated animals) are used as a further assessment. T/C values that are less than 42% are considered to be active by National Cancer Institute Standards; T/C values less than 10% are considered to have excellent activity and potential clinical activity by National Cancer Institute standard.
Vinblastine, cytochalasin B, tetramethylrhodamine lisothiocyanate (TRITC)-phalloidin, sulforhodamine B (SRB) and antibodies against xcex2-tubulin and vimentin are commercially available from recognized commercial vendors. Basal Medium Eagle containing Earle""s salts (BME) and Fetal Bovine Serum (FBS) are also commercially available.
The Jurkat T cell leukemia line and A-10 rat aortic smooth muscle cells are obtained from the American Type Culture Collection and are cultured in BME containing 10% FBS and 50 xcexcg/ml gentamycin sulfate. Human ovarian carcinoma cells (SKOV3) and a sub-line which has been selected for resistance to vinblastine (SKVLB1) were a generous gift from Dr. Victor Ling of the Ontario Cancer Institute. Both cell lines are maintained in BME containing 10% FBS and 50 xcexcg/ml gentamycin sulfate. Vinblastine is added to a final concentration of 1 xcexcg/ml to SKVLB1 cells 24 hours after passage to maintain selection pressure for P-glycoprotein-overexpressing cells.
Cell proliferation assays are performed as described by Skehan et al. For Jurkat cells, cultures are treated with the indicated drugs as described in Skehan and total cell numbers are determined by counting the cells in a hemacytometer. The percentage of cells in mitosis are determined by staining with 0.4% Giemsa in PBS followed by rapid washes with PBS. At least 1000 cells per treatment are scored for the presence of mitotic figures and the mitotic index is calculated as the ration of the cells with mitotic figures to the total number of cells counted.
A-10 cells are grown to near-confluency on glass coverslips in BME/10% FBS. Compounds in PBS are added to the indicated final concentrations and cells are incubated for an additional 24 hours. For the staining of microtubules and intermediate filaments, the cells are fixed with cold methanol and incubated with PBS containing 10% calf serum to block nonspecific binding sites. Cells are then incubated at 37xc2x0 C. for 60 min. With either monoclonal anti-xcex2-tubulin or with monoclonal anti-vimentin at dilutions recommended by the manufacturer. Bound primary antibodies are subsequently visualized by a 45 minute incubation with fluorescein-conjugated rabbit antimouse IgG. The coverslips are mounted on microscope slides and the fluorescence patterns are examined and photographed using a Zeiss Photomicroscope Ill equipped with epifluorescence optics for fluorescein. For staining of microfilaments, cells are fixed with 3% paraformaldehyde, permeabilized with 0.2% Triton X-100 and chemically reduced with sodium borohydride (1 mg/ML). PBS containing 100 nM TRITC-phalloidin is then added and the mixture is allowed to incubate for 45 min. At 37xc2x0 C. The cells are washed rapidly with PBS before the coverslips are mounted and immediately photographed as described above.
Dose-response curves for the effects of cryptophycin compounds and vinblastine on cell proliferation and the percentage of cells in mitosis are determined.
Aortic smooth muscle (A-10) cells are grown on glass coverslips and treated with PBS, 2 xcexcM cytochalasin B, 100 nM vinblastine or 10 nM cryptophycin compounds. After 24 hours, microtubules and vimentin intermediate filaments are visualized by indirect immunofluorescence and microfilaments are stained using TRITC-phalloidin. The morphological effects of each drug is examined. Untreated cells displayed extensive microtubule networks complete with perinuclear microtubule organizing centers. Vimentin intermediate filaments were also evenly distributed throughout the cytoplasm, while bundles of microfilaments were concentrated along the major axis of the cell. Cytochalasin B caused complete depolymerization of microfilaments along with the accumulation of paracrystalline remnants. This compound did not affect the distribution of either microtubules or intermediate filaments. The cryptophycin treated microtubules and vimentin intermediates are observed for depletion of microtubules, and collapse of rimentin intermediate filaments.
A-10 cells are treated for 3 hours with 0 or 10 xcexcM taxol before the addition of PBS, 100 nM vinbiastine or 10 nM cryptophycin compound. After 24 hours, microtubule organization is examined by immunofluorescence as described above. Compared with those in control cells, microtubules in taxol-treated cells were extensively bundled, especially in the cell polar regions. As before, vinblastine caused complete depolymerization of microtubules non-pretreated cells. However, pretreatment with taxol prevented microtubule depolymerization in response to vinblastine. Similarly, microtubules pretreated with taxol are observed with cryptophycin treatment.
A-10 cells are treated with either 100 nM vinblastine or 10 nM cryptophycins for 24 hr., resulting in complete microtubule depolymerization. The cells are then washed and incubated in drug-free medium for periods of 1 hour or 24 hours. Microtubules repolymerized rapidly after the removal of vinblastine, showing significant levels of microtubules after 1 hour and complete morphological recovery by 24 hour. Cells are visualized for microtubule state after treatment with a cryptophycin compound of this invention at either 1 hour or 24 hours after removal of the cryptophycin compounds.
SKOV3 cells are treated with combinations of cryptophycins and vinblastine for 48 hours. The percentages of surviving cells are then determined and the IC50s for each combination is calculated.
SKVLB1 cells are resistant to natural product anticancer drugs because o their over expression of P-glycoprotein. The abilities of taxol, vinblastine and cryptophycin compounds to inhibit the growth of SKOV3 and SKVLB1 cells are observed. Taxol caused dose-dependent inhibition of the proliferation of both cell lines with IC50s for SKOV3 and SKVLB1 cells of 1 and 8000 nM, respectively. Vinblastine also inhibited the growth of both cell lines, with IC50s of 0.35 and 4200 nM for SKOV3 and SKVLB1 cells, respectively. Cryptophycins compounds of this invention demonstrate activity with an IC50s of from about 1 to about 1000 pM for SKOV3 and SKVLB1 cells.
Thus, it can be demonstrated that the present invention provides novel cryptophycin compounds which are potent inhibitors of cell proliferation, acting by disruption of the microtubule network and inhibition of mitosis. These studies can illustrate that cryptophycin compounds disrupt microtubule organization and thus normal cellular functions, including those of mitosis.
Classic anti-microtubule agents, such as colchicine and Vinca alkaloids, arrest cell division at mitosis. It seems appropriate to compare the effect of one of these agents on cell proliferation with the cryptophycin compounds. For this purpose, the Vinca alkaloid vinblastine was selected as representative of the classic anti-microtubule agents. Accordingly, the effect of cryptophycin compounds and vinblastine on the proliferation and cell cycle progression of the Jurkat T-cell leukemia cell line is compared.
Since antimitotic effects are commonly mediated by disruption of microtubules in the mitotic spindles, the effects of cryptophycin compounds on cytoskeletal structures are characterized by fluorescence microscopy. Immunofluorescence staining of cells treated with either a cryptophycin compound or vinblastine demonstrate that both compounds cause the complete loss of microtubules. Similar studies with SKOV 3 cells can show that the anti-microtubule effects of cryptophycin compounds are not unique to the smooth muscle cell line.
Selected wells of a 96 well plate were seeded with GC3 human colon carcinoma cells (1xc3x9710 cells in 100 xcexcl assay medium/well) twenty four hours prior to test compound addition. Cell free assay medium was added to other select wells of the 96 well plate. The assay medium.(RPMI-1640 was the medium used; however, any medium that will allow the cells to survive would be acceptable) was supplemented with 10% dialyzed fetal bovine serum and 25 mM HEPES buffer.
The test compound was stored in an amber bottle prior to testing. Fresh dimethylsulfoxide stock solution (200 xcexcg/ml) was prepared immediately prior to preparation of test sample dilutions in phosphate-buffered saline (PBS). A dilution of 1:20 dimethylsulfoxide solution in PBS was prepared such that the final concentration was 10 ug/ml. Serial 1:3 dilutions using PBS (0.5 ml previous sample of 1 ml PBS) were prepared. Falcon 2054 tubes were used for the assay.
A 10 ul sample of each dilution of test compound was added in triplicate to wells of GC3 plates. The plates were incubated for 72 hours at about 37xc2x0 C. A 10 xcexcl sample of stock 3-[4,5-dimethyl-2-yl]-2,5-diphenyltetrazolium bromide salt (xe2x80x9cMTTxe2x80x9d 5 mg/ml in PBS) was added to each well. The plates were incubated for about an hour at 37xc2x0. The plates were centrifuged, media was decanted from the wells and 100 xcexcl acid-isopropanol (0.04 N HCl in isopropanol) was added to each well. The plate was read within one hour using a test wavelength of 570 nm (SpectraMax reader).
Evaluation of compounds of Formula I suggest that the compounds can be useful in the treatment methods claimed herein. Further, the compounds will be useful for disrupting the microtubule system.
The preparation of the compounds of this invention can be completed using several protocols involving an activated ester followed by chromatography and acid induced deblocking where necessary. 
For example, the treatment of 1(wherein the bolded numbers refer to the compound numbers indicated in the Example section) with acetic anhydride in the presence of triethylamine and 4-dimethylamino pyridine provides 3 in 89% yield after flash chromatography. Similarly, 4 is prepared from 1 via the agency of succinic anhydride followed by reverse phase HPLC purification. Exposure of pyridine solution of 1 to commercially available nicotinoyl chloride hydrochloride in the presence of triethylamine and 4-dimethylamino pyridine followed by chromatogrpahy and hydrogen chloride treatment gives rise to 7 in high yield. Pyridinium salt 8 is prepared in 47% yield according to the method of Nicolaou, Angew. Chem. Int. Ed. Engl., 1994, 33 whereby 1 is treated with commercially available 2-fluoro-1-methylpyridinium p-toluenesulfonate followed by reverse phase HPLC purification with concomitant anion exchange (acetate for p-toluenesulfonate) and lyophilization. Esters 5, 9, 11, 13, 15, 17, 19, and 21 are all prepared in moderate to high yields from 1 and commercially available (except in the case of 5 and 9) N-t-boc protected amino acids with activation via the agency 1,3-dicyclohexylcarbodiimide in the presence of 4-dimethylamino pyridine. Hydrochloride salts 10, 12, 14, 16, 18, 20 and 22 are prepared in high yield from 9, 11, 13, 15, 17, 19, and 21 respectively upon treatment with a 4.0 M solution of hydrogen chloride in dioxane and removal of solvent in 25 vacuo. Di-sodium salt 6 is derived from 5 following hydrochloric acid induced t-butylester cleavage and sodium hydroxide treatment. The requisite acid 24 for the preparation of 5 is synthesized in 63% yield by way of a 5 step sequence featuring the method of Johns, Synthesis, 1988, 142, for installing the phosphate functionality. 
Several of the novel conjugates have been assayed for in vitro cytotoxicity in the GC3 tumor cell model. From the results depicted in Table 2 it is clear that good to excellent activity relative to that of cryptophycin 55 (1) is inherent to this series of compounds.
Preparation of any ester of type 2 (R1 or R2 derived from a carboxylic acid) includes a variety of technologies employing acid chlorides, anhydrides, and common activating reagents (e.g., carbodiimides).2 Any solvent other than participating alcohols can be used. Any mild bases and/or catalysts (amines, carbonates) can be used to aid in esterification.
The conversion of carbamates 9, 11, 13, 15, 17, 19, and 21 to the corresponding salts could be effected with any strong acid, namely, mineral acids comprised of hydrogen halides, hydrogen sulfates, hydrogen phosphates, hydrogen nitrates, hydrogen perchlorates, or strong organic acids such as trifluoroacetic, p-toluenesulfonic, and methanesulfonic. The same acids could be used to produce salts of type 7 from the corresponding free base. A variety of counterions (cations) could comprise salts of type 6 including any of the alkali and alkaline earthxe2x80x2 metals. A variety of counterions (anions) could comprise salts of type 8, namely, any conjugate base of an acid (organic or mineral).
Table 2. In vitro cytotoxicity data for cryptophycin derivatives using the assay described supra.
Additional compounds of this invention and the GC3 assay results are indicated:
Additional compounds of this invention have been tested in the GC3 assay and had IC50 values ranging from less than one to about 700 nM; however, the most typical values were less than 100 nM.
Results from comparative preliminary solubility studies among 27, 1, and 12 are shown in Table 3. These results indicate enhanced solubility of 12.
A comparison of stability characteristics between 1 and 12 in aqueous Ph ranging from 4-8 was determined at room temperature for 6 hours. Results from these studies clearly demonstrated the superior aqueous solubility and stability profile of 12. For example, at pH 8, Cryptophycin 55 had a solubility value of 10 mg/mL at room temperature compared to a solubility of 30 mg/mL for 12 under substantially the same conditions. At a pH of 4, Cryptophycin 55 had an aqueous solubility value of about 50 mg/mL; however, the aqueous solubility of Cryptophycin 55 declined as the pH became basic, while the aqueous solubility of 12 remained substantially steady over the pH range studied.
Based on results from solubility and stability studies, appropriate parenteral vehicles were chosen to determine absolute solubility/stability characteristics of 12. Table 4 illustrates the solubility profile of 12 in these vehicles. Preliminary results indicate acceptable stability of 12 in formulation 6 for up to 3 weeks.
Thus, it is feasible to achieve high concentrations of a compound of this invention in a vehicle containing no surfactant or an emulsifier which in turn should enable facile toxicological and clinical evaluations of these compounds. The glycinate ester (12) also affords better stability in an aqueous environment in physiological pH range over longer periods of time indicating enhanced shelf-life under normal conditions. Whereas it is necessary to prepare concentrates of 1 and 27 for storage and then diluted prior to administration, it is feasible to prepare ready to use solutions of compounds of this invention.
Compounds of Formula I can be prepared using a compound of the formula II 
wherein
Ar, R1, R2, R3, R4, R5, R7, R8, R9, R10 have the meanings set for supra in Formula I.
R13 is selected from the group consisting of t-butylcarbamate (BOC);
R24 is selected from the group consisting of 
xe2x80x83(N-hydroxysuccinimide, herein xe2x80x9cNHSxe2x80x9d), N-hydroxysulfosuccinimide and salts thereof, 2-nitrophenyl, 4-nitrophenyl, and 2,4-dichlorophenyl;
X is O, NH or alkylamino;
Y is O, NH, or alkylamino.
Compounds of Formula III 
wherein the R groups and various substituents are as defined hereinbefore and throughout the specification; can be prepared by contacting a compound of the formula IV 
R25 is
with an acid of the formula 
R27 is selected from the group consisting of H, C1-C12 alkyl, and aryl;
and a silylating agent. Bis N,O-trimethylsilyl acetamide (BSA) is an especially preferred silylating agent.
As used herein, the phrase xe2x80x9cactive ester substituentxe2x80x9d refers to a substituent which makes the indicated substituent a good leaving group. Appropriate substituents can be selected with guidance from standard reference guides, for example, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, (London and New York, 1973); Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981). See especially pages 180 through 184 of Greene. An especially preferred active ester substituent group is N-hydroxy-succinimide. (NHS) Other preferred groups include, but are in no way limited to: 
(O-N-hydroxysuccinimide), O-N-hydroxysulfosuccinimide and salts thereof, O-2-nitrophenyl, O-4-nitrophenyl, and O-2,4-dichlorophenyl, wherein the xe2x80x9cOxe2x80x9d refers to the oxygen group necessary to form the ester functionality.
As used herein the term xe2x80x9camidexe2x80x9d refers to an amide functionality that can be cleaved using alkaline conditions. For example, the term refers to but is in no way limited to, xe2x80x94NMe2. For additional guidance, see for example Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981).
As used herein the phrase xe2x80x9cactive ester substituentxe2x80x9d refers to a substituent which makes the OR24 substituent a good leaving group. Appropriate substituents can be selected with guidance from standard reference guides, for example, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, (London and New York, 1973); Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981). An especially preferred R25 group is N-hydroxy-succinimide. (NHS)
The processes described herein are most preferably completed in the presence of a solvent. The artisan can select an appropriate solvent for the above described process. Inert organic solvents are particularly preferred; however, under certain conditions an aqueous solvent can be appropriate. For example, if R27 is hydrogen and R13 is BOC an aqueous base as solvent will be effective.
When the desired R6 substituent in the compound of Formula I contains an amine, then the amine substituent of the R6 group must be protected using an amino protecting group. The artisan can readily select an appropriate amino protecting group using guidance from standard works, including, for example, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, (London and New York, 1973); Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981).
R27 should be a group that allows for the removal of the xe2x80x94CO2R27 substituent using acidic, neutral, or mild basic conditions. Preferred R27 groups include, but are in no way limited to, hydrogen, C1-C6 alkyl, tricholoromethyl, trichloroethyl, and methylthiomethyl. It is especially preferred that R27 is hydrogen.
To provide further guidance for the artisan, the following schemes are provided:
Compounds of Formula I can be prepared using a compound of the formula II 
wherein
Ar R1, R2, R3, R4, R5, R7, R8, R9, R10 have the meanings set for supra in Formula I. The term R11xe2x80x2 is as defined for R11, supra.
R13 is a selected amino protecting group;
R24 is selected from the group consisting of active ester substituent, amide substituent, O-2-nitrophenyl, O-4-nitrophenyl, and O-2,4-dichlorophenyl;
X is O, NH or alkylamino;
Y is C, O, NH, S, SO, SO2 or alkylamino.
Compounds of Formula III 
wherein the R groups and various substituents are as defined hereinbefore and throughout the specification; can be prepared by contacting a compound of the formula IV 
R25 is
with an acid of the formula 
R27 is selected from the group consisting of H, C1-C12 alkyl, and aryl;
and a silylating agent. Bis N,O-trimethylsilyl acetamide (BSA) is an especially preferred silylating agent.
As used with regard to R25 the phrase xe2x80x9cactive ester substituentxe2x80x9d refers to a substituent which makes the OR24 substituent a good leaving group. Appropriate substituents can be selected with guidance from standard reference guides, for example, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, (London and New York, 1973); Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981). An especially preferred R25 group is N-hydroxy-succinimide. (NHS)
The processes described herein are most preferably completed in the presence of a solvent. The artisan can select an appropriate solvent for the above described process. Inert organic solvents are particularly preferred; however, under certain conditions an aqueous solvent can be appropriate. For example, if R27 is hydrogen and R13 is BOC an aqueous base as solvent will be effective.
When the desired R6 substituent in the compound of Formula I contains an amine, then the amine substituent of the R6 group must be protected using an amino protecting group. The artisan can readily select an appropriate amino protecting group using guidance from standard works, including, for example, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, (London and New York, 1973); Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981).
R27 should be a group that allows for the removal of the xe2x80x94CO2R27 substituent using acidic, neutral, or mild basic conditions. Preferred R27 groups include, but are in no way limited to, hydrogen, C1-C6 alkyl, tricholoromethyl, trichloroethyl, and methylthiomethyl. It is especially preferred that R27 is hydrogen.
To provide further guidance for the artisan, the following schemes are provided: 
As used in Scheme Ixe2x80x2 and throughout the specification, R1xe2x80x2 is halogen, SH, amino, monoalkylamino, dialkylamino, trialkylammonium, alkylthio, dialkylsulfonium, sulfate, phosphate or a. protected OH or protected SH group; R2 is OH or SH; R26 is an alcohol protecting group introduced during a portion of the synthetic process to protect an alcohol group which might otherwise react in the course of chemical manipulations, and is then removed at a later stage of the synthesis. Numerous reactions for the formation and removal of such a protecting groups are described in a number of standard works, including, for example, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d, Plenum Press, (London and New York, 1973); Greene, T. W. xe2x80x9cProtecting Groups in Organic Synthesisxe2x80x9d, Wiley (New York, 1981). The skilled artisan can select an appropriate alcohol protecting group particularly with guidance provided from such works. One particularly useful alcohol protecting group is tert-butyldimethylsilyl (TBS). The products of such schemes can be derivatized using standard methods to provide other cryptophycin compounds. 
In general terms processes of this invention are as illustrated below: 
R6 and R11xe2x80x2 is as defined herein throughout the specification.
The product of the schemes provided herein can be further derivatized using standard methods to provide further cryptophycin compounds.
The artisan can utilize appropriate starting materials and reagents to prepare desired compounds using the guidance of the previous schemes and following examples.
The ester starting material can be prepared, for example, as follows: 
R6 has the meaning defined supra.
To provide further guidance, the following schemes are provided. Certain abbreviations are used in the Schemes, Preparations and Examples which are generally known in the art. For convenience, these abbreviations include:
DMAP 4-dimethylaminopyridine
BOC tert-butoxycarbonyl
mcpba m-chloroperbenzoic acid
TMSCl chlorotrimethylsilane
HEW Horner-Emmons-Wadsworth reaction (standard reaction for olefination of an aldehyde using a phosphonate and a base)
TMG 1,1,3,3-tetramethylguanidine (standard base used for the HEW reaction)
DIBAL diisobutylaluminum hydride (standard reagent for the reduction of an unsaturated ester to an allylic alcohol)
SAE Sharpless Asymmetric Epoxidation (established reaction for the enantioselective epoxidation of allylic alcohols)
TBS tert-butyldimethylsilyl
TBS-Otf TBS trifluoromethanesulfonate (standard reagent for the t-butyldimethylsilylation of alcohols)
AIBN 2,2xe2x80x2-azobis(isobutyronitrile) (standard radical initiator)
ACN acetonitrile
DBU 1,8-diazabicyclo[5.4.0]undec-7-ene (standard amine base)
EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide
The scheme for preparing the ester is further explained by the Preparation Section herein which provides one specific application of the scheme for the convenience of the skilled artisan. 
the Ar substituents claimed herein. The scheme illustration is not intended to limited the synthesis scheme only to the phenyl ring illustrated. Rather, the artisan can broadly apply this process to provide desired starting materials for the compounds claimed herein.
The necessary reaction time is related to the starting materials and operating temperature. The optimum reaction time for a given process is, as always, a compromise which is determined by considering the competing goals of throughput, which is favored by short reaction times, and maximum yield, which is favored by long reaction times.
Certain compounds of this invention can be prepared using a biosynthetic route. For example, a solution of cryptophycin 3 (about 0.24 mmol) in DME-H2O (2:1, 15.0 mL) at about 0 C is treated with bromosuccinimide (about 65 mg) and allowed to warm to room temperature. After about 24 hours the solvent is removed and the residue dissolved in a solvent such as CH3CN and subject to reverse phase HPLC (65:35 CH3CN/H2O, 6 mL/min is preferred) to give a mixture of cryptophycins. The mixture is rechromatographed by normal phase HPLC (1:1 hexane/ethyl acetate, 3 mL/min preferred) to give the desired cryptophycins.
Likewise, cryptophycin 3 (about 40 mg, 0.063 mmol) is put into 2:1 DME/H2O (about 6 mL) and N-chlorosuccinimide (0.075 mmol) is added and is heated at about 60-70 C for about 24 hours. A further quantity of NCS (8 mg) is added and continue heating for about another 24 hours. The solvent is removed and the residue subject to HPLC (35% H2O/CH3CN 6 mL/min) to give the desired cryptophycin compounds. The resulting mixture is further purified by HPLC using methanol/water (about 4:1) to obtain the desired purified cryptophycin compounds.
To further illustrate the invention the following examples are provided. The scope of the invention is in no way to be construed as limited to or by the following examples.
Step 1. Methyl 5-Phenylpent-2(E)-enoate. A solution of trimethyl phosphonoacetate (376 g, 417 mL, 2.07 mol) in THF (750 mL) was stirred at 0xc2x0 C. in a 3 L 3-neck round bottom flask equipped with a mechanical stirrer and N2 inlet. To the chilled solution, neat tetramethyl guanidine (239 g, 260 mL, 2.07 mol) was added dropwise via an addition funnel. The chilled clear pale yellow solution was stirred for 25 minutes at 0xc2x0 C. A solution of hydrocinnamaldehyde (90%, 253 g, 248 mL, 1.9 mol) in THF (125 mL) was added dropwise to the reaction solution slowly. Upon completion of addition, the reaction was stirred for 10 h rising to room temperature. GC indicated a 95:5 ratio of product to starting material. 500 ml of water was added to the reaction vessel and the reaction stirred overnight separating into two layers. The organic layer was isolated and the aqueous layer was extracted with t-BuOMe. The organic layers were combined and dried over MgSO4, then concentrated in vacuo to yield an orange oil. The crude product was distilled at 129xc2x0 C./0.3 mm Hg yielding 360.5 g, 91.7% yield, of a clear slightly yellow oil.
EIMS m/z 190 (13; M+), 159 (410, 158 (39), 131(90), 130(62), 117 (22), 104 (12), 95 (57), 91 (100), 77 (21), 65 (59); HREIMS m/z 190.0998 (C12H14O2 D xe2x88x920.4 mnu); UV lmax (e) 210 (8400), 260 (230) nm; IR nmax 3027, 2949, 1723, 1658, 1454, 1319, 1203, 978, 700 cmxe2x88x921; 1H NMR d (CDCl3) 7.15-7.3 (Ph-H5; bm), 7.00 (3-H; dt, 15.6/6.6), 5.84 (2-H; dt, 15.6/1.2), 3.70 (OMe; s), 2.76 (5-H2; t, 7.2), 2.51 (4-H2; bdt, 6.6/7.2); 13C NMR d (CDCl3) 166.9 (1), 148.3 (3), 140.6 (Ph-11xe2x80x2), 128.4/128.2 (Ph2xe2x80x2/3xe2x80x2/5xe2x80x26xe2x80x2), 126.1 (Ph 4:), 121.4 (2). 51.3 (OMe), 34.2/33.8 (4/5).
Step 2. 5-phenyl-pent-2-en-1-ol. To a 12 L 4-neck round bottom flask equipped with a thermocouple, mechanical stirrer and N2 inlet, a solution of enoate ester (310.5 g, 1.5 mol) in THF (1.5 L) was charged and chilled to xe2x88x9271 xc2x0 C. via a i-PrOH/CO2 bath. To the reaction vessel, was added dropwise DIBAL (2.5 L, 1.5 M in toluene, 3.75 mol) at a rate to maintain the reaction temperature less than xe2x88x9250xc2x0 C. Upon complete addition, the reaction was stirred overnight with the reaction temperature less than xe2x88x9250xc2x0 C. TLC (3:1 Hexanes:EtOAc, SiO2) indicated absence of starting material after 16 h. The reaction temperature was allowed to raise to xe2x88x9215xc2x0 C. The reaction was quenched slowly with 1N HCl (150 mL). At this point the reaction setup into a gelatinous solid. A spatula was employed to breakup the semi-solid and 1N HCl (200 mL) was added making the mixture more fluid. Concentrated HCl (625 mL) was charged to form a two phase system. The layers were separated and the product extracted with t-BuOMe. The organic layer was dried over MgSO4 and concentrated in vacuo to yield a clear pale yellow oil, 247.8 g. The crude product was distilled at 145xc2x0 C./0.25 mm Hg yielding 209.7 g, 86.2%.
EIMS m/z 162 (1: M+) 144 (16), 129 (7), 117 (9) 108 (6), 92 (17), 91 (100), 75 (5), 65 (12), HREIMS m/z 162, 1049 (C11H14O, D xe2x88x920.4 mmu); UV lmax (e) 206 (9900), 260 (360); IR nmax 3356, 2924, 1603, 1496, 1454, 970, 746, 700 cmxe2x88x921; 1H NMR d 7.15-7.3 (Ph-H5; m), 5.70 (3-H; dt, 15.6/6.0), 5.61 (2-H; dt, 15.6/4.8), 4.02 (1-H2; d 4.8), 2.68 (5-H2; t, 7.2), 2.40 (OH; bs), 2.36 (4-H2; dt, 6.0/7.2); 13C NMR d141.6 (Ph 1xe2x80x2), 131.8 (3), 129.5 (2), 128.3/128.2 (Ph 2xe2x80x2/3xe2x80x2/5xe2x80x2/6xe2x80x2), 125.7 (Ph 4xe2x80x2), 63.3 (1), 35.4/33.8 (4/5).
Step 3. (2S,3S)-2,3-Epoxy-5-phenyl-1-pentanol. To a 1 L 3 neck round bottom flask equipped with a mechanical stirrer, thermocouple and nitrogen inlet was added CH2Cl2 (350 mL), dried 4 Amolecular sieves (30 g) and L-(+)-diethyl tartrate (7.62 g, 0.037 mol). The resulting mixture was cooled to xe2x88x9220xc2x0 C. and treated with Ti(O-i-Pr)4 (9.2 mL, 0.031 mol), followed by the addition of t-butylhydroperoxide (4.0 M in CH2Cl2, 182 mL, 0.78 mol) at a rate to maintain the temperature 2 xe2x88x9220xc2x0 C. Upon complete addition, the reaction mixture was stirred for another 30 min, and then treated with a solution of the allylic alcohol (50 g, 0.31 mol) in CH2Cl2 (30 mL) at a rate to maintain the temperature 2 xe2x88x9220xc2x0 C. The reaction was stirred at the same temperature for 5 h, then filtered into a solution of ferrous sulfate heptahydrate (132 g) and tartaric acid (40 g) in water (400 mL) at 0xc2x0 C. The mixture was stirred for 20 min, then transferred to a separatory funnel and extracted with t-BuOMe (2xc3x97200 mL). The combined organic phase was stirred with 30% NaOH solution containing NaCl, for 1 h at 0xc2x0 C. The layers were again separated, and the aqueous phase extracted with t-BuOMe. The combined organic phase was washed with brine, dried over MgSO4 and concentrated to yield 52.8 g as an amber oil.
Step 4. (2R, 3R)-2-hydroxy-3-methyl-5-phenylpentan-1-ol. To a 5 L 3 neck round bottom flask equipped with a mechanical stirrer, thermocouple and nitrogen inlet was added hexanes (1 L) and cooled to 0xc2x0 C. A 2.0M solution of Me3Al in hexanes (800 mL, 1.6 mol) was added, followed by a solution of the epoxide (120 g, 0.677 mol) in hexanes (250 mL)/CH2Cl2 (50 mL) maintaining the temperature below 20xc2x0 C. Upon complete addition, the cloudy reaction mixture was stirred at 5xc2x0 C. for 35 min, whereupon a solution of 10% HCl (300 mL) was added dropwise, followed by the addition of concd HCl (350 mL). The layers were separated, and the organic phase was washed with brine and dried over MgSO4. After removal of the volatiles in vacuo, 122.1 gram of an oil was obtained.
Step 5. (2R, 3R)-2-hydroxy-3-methyl-5-phenylpent-1-yl Tosylate. To a 2 L 3 neck round bottom flask equipped with a mechanical stirrer and nitrogen inlet was added the diol (58 g, 0.30 mol), dibutyltin oxide (1.5 g, 0.006 mol, 2 mol %), toluenesulfonyl chloride (57.5 g, 0.30 mol), CH2Cl2. (580 mL) #and triethylamine (42.0 mL, 0.30 mol). The resulting mixture was stirred at room temperature for 2 h (although the reaction was complete within 1 h), filtered, washed with water and dried over MgSO4. Concentration of the volatiles in vacuo afforded 104.1 gram of a slightly amber oil.
Step 6. (2R, 3R)-2-[ (tert-Butyldimethylsilyl)oxy]-3-methyl-5-phenylpent-1-yl Tosylate. A solution of the tosylate (100 g, 0.29 mol) and triethylamine (81.0 mL, 0.58 mol) in CH2Cl2 (1200 mL) was treated with neat TBS-OTf (99 mL, 0.43 mol) dropwise with continued stirring for another 20 min. The reaction was washed twice with brine, dried over MgSO4 and concentrated to dryness. The oil was dissolved in a minimal amount of hexanes and filtered over a silica pad, eluting with hexanes:EtOAc (9:1) to yield a slightly amber oil, 134 g.
Step 7. (2R,3R,5RS)-2-[(tert-Butyldimethylsilyl)oxy]-3-methyl-5-bromo-5-phenylpent-1-yl Tosylate. To a 5 L 3 neck round bottom flask equipped with a mechanical stirrer, reflux condenser and nitrogen inlet was added CCl4 (1680 mL), TBS Ts (140 g, 0.30 mol), NBS (65 g, 0.365 mol) and AIBN (16.5 g, 0.10 mol). The mixture was degassed by evacuation under full vacuum with stirring, and backfilling with nitrogen (3xc3x97). The reaction mixture was then heated to reflux, whereupon the color became dark brown. After 15 min at vigorous reflux, the reaction mixture became light yellow, and chromatographic analysis indicated the reaction was complete. After cooling to room temperature, the reaction was filtered and the filtrate concentrated to dryness. The residue was redissolved in hexanes and filtered again, and concentrated to dryness to afford 170.3 gram as an amber oil.
Step 8. (2R, 3R)-2-[(tert-Butyldimethylsilyl)oxy]-3-methyl-5-phenylpent-4(E)-en-1-yl Tosylate. To a 2 L 3 neck round bottom flask equipped with a mechanical stirrer, reflux condenser and nitrogen inlet was added a solution of the bromide (100 g, 0.186 mol) in acetonitrile (700 mL). DBU (83.6 mL, 0.557 mol) was added and the resulting dark brown solution was stirred at reflux for 15 min. After cooling to room temperature, the solvent was removed in vacuo, and the residue digested in CH2Cl2 (200 mL) and filtered through a silica pad. The volatiles were again evaporated, and the residue dissolved in EtOAc and washed with water, brine and dried over MgSO4 and concentrated to dryness. Preparative mplc (Prep 500) chromatography afforded the desired unsaturated compound (50.3 g, 60% yield over 4 steps).
Step 9. (3S, 4R)-3-[(tert-Butyldimethylsilyl)oxy]-4-methyl-6-phenylhex-5(E)-en-1-nitrile. The tosylate (50 g, 0.11 mol) was dissolved in DMSO (1 L) and treated with KCN (14.2 g, 0.22 mol) and water (25 mL), and the resulting mixture was stirred at 60xc2x0 C. under nitrogen for 18 h. After cooling to room temperature, the reaction mixture was partitioned between EtOAc (1 L) and water (1 L). The aqueous phase was extracted with EtOAc (500 mL), and the combined organic phase was washed with brine and dried over Na2SO4. Flash chromatography over silica with CH2Cl2 afforded the desired nitrile in 92% yield.
Step 10. Methyl (5S, 6R)-5-[(tert-Butyldimethylsilyl)oxy]-6-methyl-8-phenylocta-2 (E),7 (E) -dienoate. The nitrile (14.67 g, 46.5 mmol) was dissolved in toluene (200 mL) and cooled to xe2x88x9278xc2x0 C. under nitrogen. A 1.5M solution of DIBAL in toluene (37.2 mL, 55.8 mmol) was added dropwise with vigorous stirring. Upon complete addition, the cooling bath was removed and the reaction was stirred at room temperature for 1 h. The reaction mixture was carefully poured into 1N HCl and the mixture stirred at room temperature for 30 min. The layers were separated, and the organic phase was washed with a saturated aqueous solution of sodium potassium tartrate (2xc3x97), brine and dried over Na2SO4. The volatiles were removed in vacuo, and the crude pale yellow oil was used directly in the subsequent condensation.
The crude aldehyde from above was dissolved in THF (90 mL) and treated with trimethyl phosphonoacetate (9.03 mL, 55.8 mmol) and tetramethylguanidine (7.0 mL, 55.8 mmol) at room temperature under nitrogen. The reaction mixture was stirred for 16 h, then partitioned between EtOAc (200 mL) and water (100 mL). The aqueous phase was back extracted with EtOAc (100 mL), and the combined organic phase was washed with water, brine and dried over Na2SO4. The volatiles were removed in vacuo, and the crude yellow oil (17.0 g) was chromatographed over silica gel with CH2Cl2: cyclohexane (1:1 to 2:1) to afford 13.67 grams of the desired ester, 78.5%. 
Methyl ester (2.673 mmol) was dissolved in acetone and then 1N aqueous LiOH (26 mL) added at room temperature. The cloudy mixture was further diluted with acetone (20 mL) and the resulting yellow mixture stirred at room temperature for 23.5 h. The reaction was diluted with diethylether (400 mL) and the organics washed with 1N HCl (120 mL), brine (200 mL) and H2O (160 mL). The organics were dried and concentrated in vacuo to leave a yellow oil which was purified by column chromatography (gradient: 5% AcOH +20%-40% EtOAc/Hexanes) to give carboxylic acid as a yellow oil (960 mg, 100%). 1H NMR (CDCl3) d 7.38-7.19 (m, PhH5), 7.09 (ddd, J=15.2, 7.6 and 7.9 Hz, 3-H), 6.38 (d, J=16 Hz, 8-H), 6.16 (dd, J=16 and 8 Hz, 7-H), 5.85 (d, J=15.8 Hz, 2-H), 3.81-3.75 (m, 5-H), 2.49-2.37 (m, 6-H, 4-CH2), 1.12 (d, J=6.7 Hz, 6-Me), 0.91 (s, SiCMe3), 0.065 (s, SiMe), 0.068 (s, SiMe) ppm; IR u (CHCl3) 2957, 2930, 2858, 1697, 1258, 1098, 838 cmxe2x88x921; MS (FD) 360.2 (M+, 100); [a]D +87.6xc2x0 (c 10.5, CHCl3); Anal. calcd. for C21H32O3 requires: C, 69.95; H, 8.95%. Found: C, 69.19; H, 8.39%. 
To a stirred solution of carboxylic acid (2 mmol) in dry dimethylformamide (5.50 mL) was added 1-ethyl-3-(3-dimethyaminopropyl)carbodiimide (2.4 mmol) and N-hydroxysuccinimide (2.6 mmol) at room temperature. The mixture was stirred for 28 h and then diluted with EtOAc (100 mL) and washed with 1N aqueous HCl (2xc3x9750 mL), H2O (75 mL), dried and concentrated in vacuo to leave an oil. Crude product was purified by column chromatography (gradient: 5-30% EtOAc/Hexanes) to give active ester as a pale yellow oil (724 mg, 80%)
1H NMR (CDCl3) d 7.36-7.20 (m, PhH5, 3-H), 6.38 (d, J=16 Hz, 8-H), 6.14 (dd, J=16.1 and 8.0 Hz, 7-H). 6.03 (d, J=16 Hz, 2-H), 3.79 (q, J=4.3 Hz, 5-H), 2.94 (brs, CH2CH2), 2.58-2.42 (m, 6-H, 4-CH2), 1.10 (d, J=6.8 Hz, 6-Me), 0.90 (s, SiCMe3), 0.05 (s, SiMe2) ppm; IR u (CHCl3); 2957, 2931, 2858, 1772, 1741, 1648, 1364, 1254, 1092, 1069, 838 cmxe2x88x921; MS (FD) 457 (M+, 100); [a]D +71.3xc2x0 (c 10.1, CHCl3); Anal. calcd. for C25H35NO5 requires: C, 65.61; H, 7.71; N, 3.06%. Found: C, 65.51; H, 7.56; N, 3.02%. 
To a stirred solution of silyl ether (2.50 g, 5.47 mmol) in CH3CN (130 mL) was added 48% aqueous HF (15 mL) at 0 C. The solution was stirred at 0 C for 0.75 h and then at room temperature for 4 h. The reaction was diluted with diethylether (300 mL) and washed with H2O until the wash was xcx9cpH7. Organics were dried (MgSO4) and concentrated in vacuo to give a yellow residue which was recrystallized from Et2O to give alcohol as white crystals (1.46 g, 78%). 1H NMR (CDCl3) d 7.41-7.20 (m, PhH5, 3-H), 6.48 (d, J=16 Hz, 8-H), 6.15-6.07 (m, 7-H, 2-H), 3.71-3.65 (m, 5-H), 2.83 (brs, CH2CH2), 2.60-2.33 (m, 6-H, 4-CH2), 1.95 (brs, 5-OH), 1.14 (d, J=6.8 Hz, 6-Me) ppm; IR u (KBr) 3457, 1804, 1773, 1735, 1724, 1209, 1099, 1067, 1049, 975, 744, 694 cmxe2x88x921; UV (EtOH) 1max 250 (e=20535) nm; MS (FD) 343.2 (M+, 100); [a]D xe2x88x9257.8xc2x0 (c 10.56, CHCl3); Anal. calcd. for C19H21NO5S requires: C, 66.46; H, 6.16; N, 4.08%. Found: C, 66.49; H, 6.16; N, 4.07%. 